Role of HIF-1 in Iron Regulation

Current Molecular Medicine 2006, 6, 883-893 883

Role of HIF-1 in Iron Regulation: Potential Therapeutic Strategy forNeurodegenerative Disorders

Donna W. Lee and Julie K. Andersen*

Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945, USA

Abstract: A disruption in optimal iron levels within different brain regions has been demonstrated inseveral neurodegenerative disorders. Although iron is an essential element that is required for manyprocesses in the human body, an excess can lead to the generation of free radicals that can damagecells. Iron levels are therefore stringently regulated within cells by a host of regulatory proteins thatkeep iron levels in check. The iron regulatory proteins (IRPs) have the ability to sense and control thelevel of intracellular iron by binding to iron responsive elements (IREs) of several genes encoding keyproteins such as the transferrin receptor (TfR) and ferritin. Concurrently, the hypoxia-inducible factor(HIF) has also been shown in previous studies to regulate intracellular iron by binding to HIF-responsive elements (HREs) that are located within the genes of iron-related proteins such as TfR andheme oxygenase-1 (HO-1). This review will focus on the interactions between the IRP/IRE andHIF/HRE systems and how cells utilize these intricate networks to regulate intracellular iron levels.Additionally, since iron chelation has been suggested to be a therapeutic treatment for disorders suchas Parkinson’s and Alzheimer’s disease, understanding the exact mechanisms by which iron acts tocause disease and how the brain would be impacted by iron chelation could potentially give us novelinsights into new therapies directed towards preventing or slowing neuronal cell loss associated withthese disorders.

1. CELLULAR IRON HOMEOSTASIS iron-catalyzed oxidative damage. While sequesteringiron in a relatively non-reactive state, the intracellulariron pool is also decreased from participating in awide variety of cellular functions.

Iron is the most abundant metal in the brain [1]and highest levels are found within the basalganglia, which comprises the putamen, caudatenucleus, and globus pallidus [2]. Some degree ofaccessible reactive iron is necessary for brain viabilityas it serves as a cofactor in DNA, RNA, and proteinsynthesis, for heme and non-heme enzymesinvolved in both mitochondrial respiration andneurotransmitter synthesis [3,4]. While irondeficiencies early in life are known to result inimpairments in brain development [5], highconcentrations of iron may result in cellular toxicity[6], due to its ability to catalyze the production ofreactive oxygen species. Therefore, cells continuallymonitor and regulate the available pool of labile ironto limit adverse reactions of iron with otheroxygen/nitrogen radicals [7].

Cellular iron homeostasis is maintained by theconcerted binding actions of iron regulatory proteins(IRPs) 1 and 2 to the iron-responsive elements(IREs; 5’-CAGUGU/C-3’) located on the 3’ end of thetransferrin receptor (TfR) and divalent metaltransporter-1 (DMT1) mRNAs and 5’ end of theferritin (Ft) and mitochondrial (m)-aconitase mRNAs[9]. IRPs sense intracellular iron levels and respondaccordingly by modifying the expression of proteinsinvolved in uptake (e.g. TfR) and storage (e.g. Ft).When iron levels are low, IRP binding to IREslocated in the 5’ UTR of transcripts of Ft isenhanced, thereby effectively preventing theinitiation of translation of the iron storage protein.Additionally, IRP binding to 3’UTR of TfR and DMT-1mRNAs protects them from being degraded, resultingin an up-regulation of iron uptake into the cell byreceptor-mediated endocytosis and its intracellularrelease. Conversely, when intracellular iron levels arehigh, IRPs dissociate from IREs, resulting in thebreakdown of TfR and DMT-1 and up-regulation of Ftand m-aconitase to sequester cellular iron [9].

The uptake of iron into most cells occurs via thereceptor-mediated endocytosis of iron-loadedtransferrin (Tf) binding to the cell surface TfR (Fig. 1).Acidification of the endosomes promotes the releaseof iron from the Tf-TfR complex and transported intothe cytosol for its utility via the divalent metaltransporter-1 (DMT-1) [8]. The resultant labile ironpool (LIP) is then sequestered by storage proteins,used for synthesis of iron-containing proteins, andcatalysis of enzymatic and/or oxidative reactions.Ferritin is the main iron storage protein of the brainand is a key component in the protection against

IRP1 is a 98kDa protein that shares highhomology with m-aconitase which converts citrate toisocitrate in the tricarboxylic acid (TCA) cycle. Thepost-translational switch of IRP1 between anapoprotein and a holoprotein is dependent on theassembly of the [4Fe-4S] cluster, which is highlyregulated by intracellular iron levels [10]. Theconformational structure of the domains of the

*Address correspondence to this author at the Buck Institute for AgeResearch, 8001 Redwood Blvd., Novato, CA 94945, USA; Tel: +1 (415)209-2070; E-mail: [email protected]

1566-5240/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

884 Current Molecular Medicine, 2006, Vol. 6, No. 8 Lee and Andersen

Fig. (1). A simplified schematic of the iron regulatory system adapted from [133]. Ferric iron is uptaken into the cell viareceptor-mediated endocytosis of the TfR. Following conversion to ferrous iron within acidified endosomes, iron istransported via the DMT-1 into the cytosolic labile iron pool (LIP). The available iron is then distributed for variousfunctions, such as activation of IREs, sequestration by MT-III or ferritin for future use, participation in iron-sulfur clustersynthesis or iron-catalyzed reactions by PANK2 within mitochondria, as well as cellular efflux via the IREG1. (CO, carbonmonoxide; BV, biliverdin; CP, ceruloplasmin, DMT-1, divalent metal transporter-1, Hep, hephaestin; PANK2, pantothenatekinase 2).

holoprotein allows for the assembly of the [4Fe-4S]cluster coordinated by cysteine residues allowing forenzymatic aconitase activities in the cytosol [11]. Onthe other hand, the disassembly of the [4Fe-4S]cluster essentially creates space for IREs to bind tothe apoprotein [12]. When cellular iron levels are low,the RNA binding capacity of IRP1 is elevated toenhance the synthesis of the TfR and suppress thesynthesis of ferritin, ensuring that the cellular needfor iron is met. When iron levels increase to allow forcluster assembly, cytosolic (c)-aconitase activity isreconstituted [13].

2. IRON DYSREGULATION IN NEURO-DEGENERATIVE DISORDERS

A disruption in iron homeostasis has beendemonstrated in models of neurodegeneration[16,17] and hypoxia-ischemia [18]. Although brainiron concentration normally increases with age inrodents [19] and humans [20], the accumulation ofiron in patients with neurodegenerative disorders iseven more elevated, suggesting that an excess ofiron is involved in neuronal damage. However, therehave been few definitive studies to prove thehypothesis that iron promotes neurodegenerationand other studies have suggested that iron may beaccumulating as a result of neuronal cell loss.

IRP2 is highly homologous with IRP1 with theexception of the presence of an additional 73-aminoacid motif and a lack of an iron-sulfur cluster [14]. Iniron-replete or normoxic cells, the rapid degradationof IRP2 by the proteasome is dictated, in part, byiron-dependent oxidative modifications to thecysteine and proline residues within the 73-aminoacid motif [14]. Much like IRP1, the RNA bindingcapacity of IRP2 is enhanced when intracellular ironlevels are low. But unlike IRP1, the activation ofIRP2 requires de novo synthesis of the protein [15].

2a. Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerativedisorder that stems from the loss of dopaminergic(DAergic) neurons within the substantia nigra (SN). Alack of evidence for heritability in the sporadicidiopathic form of PD has pointed toward theinvolvement of environmental risk factors in disease

Role of HIF-1 in Iron Regulation Current Molecular Medicine, 2006, Vol. 6, No. 8 885

etiology [21]. The existing elevated levels of inherentreactive oxygen species produced during cellulardopamine breakdown by monoamine oxidases or viaits auto-oxidation to quinones predisposes DAergicneurons to oxidative damage [22,23]. Additionally,hydrogen peroxide is also generated duringdopamine synthesis by tyrosine hydroxylase [24]. Ifnot properly buffered, hydroxyl radicals can stimulatelipid peroxidation which can eventually lead tomacromolecular injury and neuronal death. Some ofthe most intriguing evidence suggestive of a role foriron dysregulation in the development ofneurodegenerative disorders has been provided byseveral studies that demonstrate selective DAergiccell loss correlating with elevated levels ofintracellular iron in Parkinsonian brains [25-28].Despite an accumulation of iron in the SNpc of PDpatients, ferritin subunits are either heavily saturatedwith iron [29] or its levels are lowered [30,31],resulting in an excess of labile iron. Interestingly,IRP1 binding has also been reported to besustained in the midbrain of PD patients [32],resulting in increased TfR levels bringing more ironinto the cells. The presence of unsequestered ironserves mainly to reduce hydrogen peroxide tohydroxyl radicals via the Fenton reaction [33] and tosecondarily promote the auto-oxidation of DA [34]and formation of neuromelanin, which can sequesterFe3+ [35]. Iron can also facilitate the aggregation ofα-synuclein [36], found within intra-cytoplasmicinclusions called Lewy bodies, which are acharacteristic hallmark in the PD brain.

suggesting additional levels of APP regulation byiron [49]. Although copper and zinc are heavilyimplicated in AD pathology [50,51], dysregulation ofiron homeostatic processes have also beenobserved including elevations in plague-associatediron [52], iron-laden ferritin [53], and alterations inthe IRE/IRP system [54,55] in the patient brains andtransgenic mouse models of AD.

2c. Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disorderthat results from demyelination within the centralnervous system. Unlike PD and AD, it chronicallyaffects young adults rather than in older individuals.The etiology of this debilitating disorder is not fullyunderstood. It is pathologically characterized by thebreakdown of the blood brain barrier allowing for theactivation and recruitment of circulating T cells,macrophages and microglia into lesion sites withinthe white matter. The subsequent neuroinflammationresults in the destruction of oligodendrocytes andaxonal processes [56]. Clinical manifestationsinclude loss of sensory, motor, and cognitivefunctions.

Enrichment of iron within the oligodendrocytes ofthe normal brain probably reflects the importanceand need to meet the metabolic demandsassociated with myelinogenesis. However, adisruption in the processes regulating iron within theoligodendrocytes and myelin would also contribute tothe production of free radicals and oxidative damagewithin these cell types. Elevated iron deposition isobserved within macrophages and extravasated redblood cells of mice with experimental allergicencephalomyelitis (EAE; the animal model of MS)[57] and in plagues surrounding lesions in MSpatients [58]. Additionally, punctate iron staining wasobserved along transected axons and neurons ofafflicted MS patients [59] suggesting that themitochondria may be a possible site of deposition.

2b. Alzheimer’s Disease

Alzheimer’s disease (AD) is a leadingneurodegenerative disorder, afflicting nearly 5 millionpersons within the U.S. alone. AD is characterizedsymptomatically by dementia and biochemically byprogressive neurodegeneration and accumulation ofneurofibrillary tangles and amyloid plaques primarilyin the forebrain and hippocampus of affectedpatients [37]. Disruptions in regulatory processeswhich normally act to combat oxidative stress resultin impairments in mitochondrial energetics [38-40]within regions of the brain that control memory,thought, and language processes resulting indisease symptomology. There exist numerous piecesof evidence to suggest that pathological irondeposition may contribute to neurodegeneration [41-43] but it remains unclear if iron promotes oxidativecell damage or is released as a by-product ofneuronal cell loss.

2d. Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomalrecessive disorder that is characterized by loss oflimb reflexes, spasticity, extensor plantar responses,and limb ataxia [60]. This common inherited form ofataxia is characterized by the early degeneration oflarge sensory neurons in the dorsal root ganglionfollowed by progressive degeneration and atrophy ofthe spinal-cerebellar tracts, cortical-spinal tracts, andlarge sensory fibers of the peripheral nerves [61].This disorder results from the reduced expression offrataxin protein as a result of either point mutationsor GAA triplet repeat expansions on the FRDA gene[62].

Insoluble beta-amyloid (Aβ) peptides are the mainconstituent of senile plagues. They are generated bythe cleavage of the larger amyloid precursor protein(APP) by secretases [44,45] and are thought tocontribute to the neurotoxicity associated with AD[46]. Mounting evidence implicates redox-activemetals such as iron and their interaction with Aβ inthe promotion of oxidative stress and Aβ deposition[47,48]. IREs have been demonstrated to be locatedon the 5’ untranslated region of the APP mRNA

Frataxin is localized to the mitochondria and isnecessary for iron-sulfur cluster assembly and thesubsequent biogenesis of iron-containing enzymes[63]. Additionally, frataxin has also beendemonstrated to store excess iron [64] and activate


thiol-containing glutathione peroxidase [65], therebypreventing metal-induced oxidative stress. Therefore,it is thought that the degeneration of sensoryneurons in FRDA results from a combination ofmitochondrial iron accumulation and deficienciesresulting from an impairment of iron-sulfur cluster-containing enzymes involved in oxidativephosphorylation [66]. More recently, the ferrioxidaseactivity, converting redox-active Fe(II) to Fe (III) wasidentified as a critical function of frataxin in a seriesof comprehensive mutational yeast studies, wherebymutations that affect the iron detoxification offrataxin resulted in elevated sensitivity of yeast cellsto oxidative stress and shortening of chronologicallife span [67].

involved in angiogenesis, apoptosis, erythropoiesis,and glycolysis as a response to reduced oxygentensions. To cover the broad range of regulatoryeffects that the HIF system has on cellular functionsis beyond the scope of this review. This review willfocus on various aspects of iron regulation of HIF-1activation and the therapeutic potential of regulatingthis pathway in neurodegenerative diseases.

3a. Prolyl Hydroxylase (PHD)

The prolyl-4-hydroxylases (PHDs) represent animportant family of enzymes that require iron as anessential cofactor. These enzymes reside incytoplasm and serve to hydroxylate proline residuessituated on various proteins including various HIFs[78]. Through a series of mutational and massspectrometric experiments, the PHDs were revealedto be the link between HIF-1α and von Hippel-Lindau (vHL) protein. Since these enzymes had strictrequirements for oxygen, iron, 2-oxoglutarate, andascorbate, the investigators utilized paradigms toinclude hypoxia and 2-oxoglutarate analogs to inhibitPHD activity and ascorbate to enhance degradationof HIF-1α [81]. The degradation of HIF-1α mediatedby vHL and PHD is a pathway which has been foundto be highly conserved - EGL-9 has been identifiedas the dioxygenase that hydroxylates prolineresidues in C. elegans [79] and in D. melanogaster[80]. Additionally, a set of three mammalian HIF-hydroxylating PHDs have been identified (PHD1,PHD2, PHD3) [87] which are distinct from thecollagen prolyl-4-hydroxylase found within theendoplasmic reticulum. These act on a set ofdifferent sequences than those present around theproline residues (Pro402 and Pro564; [81]). All threePHD isoenzymes have very similar Km values foroxygen, suggesting that the catalytic activities ofthese enzymes would not differ too greatly in cellsundergoing hypoxic or anoxic conditions [86]. Underhypoxic or iron-lacking (substrate-limiting) conditions,PHDs are prevented from hydroxylating prolineresidues of the HIF-1α protein which subsequently isnot promoted for capture by the vHL forubiquitination and degradation by the proteasome.The result of an inhibition of PHD activity is thestabilization of HIF-1α and activation of HIF-regulated genes.

3. HIF-1 AND IRON…IS THERE A RELATION-SHIP?

As outlined above, iron is necessary as a cofactorin several enzymatic processes but in excess mayresult in neurodegeneration. One of the pathways inwhich iron is emerging as a possible key componentis in the control of the damaging effects of hypoxicinjury via the transcription regulatory protein hypoxia-inducible factor (HIF). HIF is a basic helix-loop-helix(bHLH) heterodimeric transcription factor that plays amajor role in oxygen homeostatic adaptation tohypoxia [68,69]. HIF is composed of the constitutiveα and β subunits. HIF-1β (~91-94kDa) is also knownas the aryl hydrocarbon receptor nuclear translocator(ARNT) and is localized within the nucleus [70].Although there are 3 different α subunits (1α, 2α,3α), HIF-1α (~120kDa) has been extensivelycharacterized and demonstrated to be the primarytranscription factor that is regulated by hypoxia. Thestability of HIF-1α results in its accumulation in thecytosol and translocation to the nucleus where itbinds HIF-1β. Together, this nuclear HIF complex,associated with the transcriptional co-activatorsCREB-binding protein (CBP) and p300 [71], bindingto hypoxia response elements (HREs; 5’-RCGTG-3’)found on a variety of genes [72,73], including hemeoxygenase-1 (HO-1) and transferrin receptor (TfR) asshown in Figs. 2a and b.

The importance and essentiality of HIF-1 in tissuesurvival are evident by the embryonic lethality anddevelopmental abnormalities observed in mice withsystemic deletion of HIF-1α [74]. In vivo genedelivery of HIF-1α into mutant embryos rescued themfrom apoptosis and vascular defects associated withHIF-1α deficiency [75]. Using a conditional Cre/LoxPsystem, neural-specific deletion of HIF-1α was foundto result in hydrocephalus and impaired spatialmemory in adult mice [74]. The activation of this HIFpathway represents a central part in thedevelopment of pathways necessary for the properfunctioning of the brain.

3b. von Hippel-Lindau (vHL)

The vHL protein is the substrate recognitioncomponent of the E3 ligase complex thatubiquitinates and targets proteins for proteasomaldegradation [82]. The identification of an oxygen-dependent domain (ODD) on HIF-1α suggested thatit may be a target of the ubiquitin-proteasomepathway [83], providing a level of regulation of HIFactivity. Additionally, the stability of HIF-1α and HIF-mediated gene regulation had been demonstratedto be largely controlled by interactions at the ODD.Constitutive HIF-1/HRE binding and transcriptionalactivation of hypoxia-regulated genes were observed

HIF-1 and its associated proteins have beendemonstrated to be important cellular oxygensensors [76,77]. As a result, the activation of HIF-1 isan essential process in the up-regulation of genes


a

b

Fig. (2). (a) Under normoxic conditions, HIF-1α is hydroxylated by PH, ubiquitinated by vHL, which is part of the E3ubiquitin ligase complex, and targeted to the proteasome for degradation.(b) Under hypoxic or iron-lacking conditions, prolyl hydroxylases are prevented from hydroxylating proline residues of theHIF-1α protein, which subsequently does not get ubiquitinated and degraded by the proteasome. The stability of HIF-1αresults in its accumulation in the cytosol and translocation to the nucleus where it binds HIF-1β and to the hypoxiaresponse elements (HREs) found on a variety of genes, including heme oxygenase-1 (HO-1) and transferrin receptor (TfR).Additionally, IRP binding to iron responsive elements (IREs) on RNAs encoding TfR and ferritin is activated, enhancing TfRand suppressing ferritin synthesis.


in cells deficient in vHL [84]. Subsequently, it wasdiscovered that HIF-1α binding to the vHL requireshydroxylation of conserved proline residues situatedwithin the ODD [85-88] via oxygen and iron as bothhypoxia and iron chelation inhibit HIF-1αdegradation. More recently, loss of the vHL protein ina human renal cell line was shown to result inaberrant HIF-mediated activation of TfR which led toelevated uptake of Tf-bound iron [89]. The up-regulation of TfR was not accompanied bycompensatory increases in ferritin levels. Despite theelevation in intracellular iron, more iron wasassociated with the ferritin found in cells with non-functional vHL. These cells also did not exhibitincreased susceptibility to oxidative damage inducedby glutathione depletion, suggesting that thedisruption in iron homeostasis was not detrimentaland that continual activation of HIF-regulated geneslikely included those that conferred protectionagainst oxidative stress.

occurs under conditions of oxidative stress. Incontrast to IRP1, hypoxia enhances IRP2 RNAbinding in both human embryonic kidney (HEK) 293and rodent hepatoma cells by way of IRP2 proteinaccumulation [91]. The degradation of IRP2 dictatesmuch of its regulatory functions as it had previouslybeen demonstrated that IRP2 RNA binding activityoccurs when iron levels are low and is degraded iniron-replete cells [14]. Excessive iron can catalyzeoxidation and subsequent ubiquitination on the IRP2protein for targeting to the proteasome [95]. Morerecently, studies have demonstrated that theproteasomal degradation of IRP2 requires 2-oxo-glutarate-dependent oxygenases which utilize iron,oxygen, and ascorbate as essential cofactors[90,96]. Interestingly, members of the family of 2-oxo-glutarate-dependent oxygenases also includethe prolyl hydroxylases that regulate HIF-1α.Although IRP2 activation is not dependent on HIF-1,several other parallels could be drawn betweenthese two proteins, whereby both proteins arestabilized by post-translational processes duringhypoxia and treatment with desferoxamine andcobalt chloride. Additionally, both proteins are alsoubiquitinated and degraded by the proteasomeunder normoxic and iron-replete conditions [14,15].These data suggest that although the regulation ofeither protein is not entirely dependent on eachother, it is possible that similar pathways underlie themechanistic regulation of IRP2 and HIF-1α.

3c. IRP1/IRP2

Besides iron, oxygen is also a key regulator of theIRP system [90]. It was demonstrated that whilehypoxia inactivates IRP1 RNA binding activity in rathepatoma cells [91], IRP1 binding activity wasincreased in human hepatoma cells [92]. Theinvestigators of the latter study suggested that thedisparity in results may have arisen from theabundance of IRP1 and IRP2, whereby the humancell line produced much less IRP1 protein. Also, thespecies from which the cell lines originated mightpossess differing metabolic and homeostatic controlsthat allows for conflicting results. The involvement of[4Fe-4S] cluster in the hypoxic regulation of IRP1was evidenced by the iron requirement for theinactivation of IRP1 [91] as well as the observeddecrease in c-aconitase activity [92]. Recently,investigators using a neuronal cell culture systemreported that while hypoxia decreased IRP1 bindingactivity in glial cells, enhanced IRP1 binding activitywas detected in cortical neurons. This wasaccompanied by an early (3h) and late (24h)increase in ferritin synthesis in glia and corticalneurons, respectively, during the reoxygenationperiod [93]. The differential modulation of IRP1 andferritin by hypoxia in neurons and glia duringhypoxia/reoxygenation is not surprising, consideringthat these two cell populations are equipped withdifferentially vulnerable and distinct machineries tocombat oxygen-related injury.

3d. Tf/TfR

Early in vivo studies have established therelationship between oxygen and iron regulation withfindings that hypoxia resulted in higher ironabsorption as well as an up-regulation of Tf in miceand rats [97,98]. Initial hypoxia-mediated up-regulation of TfR was thought to arise solely fromenhanced IRE/IRP-1 interaction [92]. However, invitro studies using human hepatoma cell lines laterdemonstrated the relevance of oxygen and HIF-1αin the hypoxia-induced up-regulation of Tf by thediscovery of HREs flanking the Tf mRNA [99]. Thiswas complemented by simultaneous findings thathypoxia induced the expression of the TfR and theidentification of an HRE within the TfR gene by twodifferent laboratories [73,100]. It is undisputed thatpost-transcriptional control of TfR expression ismediated mainly by the IRE/IRP system; howeverexperiments utilizing DFO, an iron chelator andhypoxia mimetic known to stabilize HIF-1α levels[101], and actinomycin D, a transcriptional inhibitor[102], demonstrate that a significant level of TfRexpression is transcriptionally regulated by HIF-1activation as well. The relative contribution of theIRE/IRP and HRE/HIF systems to TfR is wellillustrated in HEK293 cells exposed to hypoxia for21h in culture [103]. The investigators observed anearly increase in iron uptake followed by a latephase decrease in iron uptake, thought to resultfrom a decrease in IRP1 and increase in IRP2activity. However, since iron uptake via TfR would be

The importance of IRP2 in neuronal regulation ofiron has recently been of more interest with thefindings that IRP2 knockout animals display aberrantiron homeostasis resulting in iron accumulation withinthe brain. Whereas adult-onset neurodegenerationwas common in IRP2 knockout mice, IRP-1 knockoutmice were found to be without any obviousphenotype [16]. It has thus been suggested thatIRP2, with its higher sensitivity to iron levels [94],may be the main modulator of iron homeostasisunder physiological conditions while IRP1 activation


up-regulated via an increase rather than a decreasein IRP1 RNA binding activity, it is possible that theelevation in iron uptake is occurring via a HIF-mediated activation of TfR transcription. Theaccumulation of intracellular iron could subsequentlybe balanced by the observed increase in ferritinsynthesis. Prolonged iron uptake however wouldlead to degradation of IRP2 and HIF-1α anddestabilization of TfR.

reoxygenation period of these hypoxic cells [104].This was accompanied by hydroxylation of prolineresidues necessary for proteasomic degradation ofHIF-1α. Perhaps, the deleterious effects associatedwith reoxygenation results from an inability of cells toinduce a potentially cytoprotective HIF response.

The protective effects of HIF-1α have been wellestablished in a number of experimental models ofoxidative stress and ischemia/reperfusion injury [105-107]. Data from these studies suggest thatprotection may arise from the elevated expression ofHRE-containing genes that compensate for thedetrimental sequelae associated with hypoxic oroxidative stress. For example, up-regulation ofproteins such as LDH and GLUT1 promotesglycolysis and reduces the excessive ROS that formas byproducts of mitochondrial glucose oxidation.HIF-mediated increases in erythropoietin and othergrowth factors such as VEGF have beendemonstrated to protect neuronal cells fromapoptosis resulting from serum and glucosedeprivation [108], as well as glutathione depletion[106]. HIF-1α has also been demonstrated tomediate either pro-death or pro-survival responses inthe same cell type, depending on the injury stimulus[109]. Hippocampal cells treated with DNA-damagingand endoplasmic reticulum stress-inducing agentswere protected from cell death by HIF-1α over-expression. In contrast, it was shown that forcedexpression of an oxygen-stabilized form of HIF-1

4. THERAPEUTIC TARGETING OF HIF-PHWith increasing evidence of the involvement of

HIF-regulated genes encoding proteins involved iniron regulation, cell survival, apoptosis, proliferation,and energy metabolism (Table 1), the iron-dependent PHDs have been implicated as targetsfor neuroprotection in the central nervous system.However, the neuroprotective and/or detrimentaleffects of HIF-1α activation appear to be dependentupon the duration and intensity of protein inductionas well as the specific cellular microenvironment.Although this has not been demonstrated in a widevariety of animal models, cells are apparently alsoequipped with feedback mechanisms to ensure thatthe activity of PHD is not suppressed for extendedperiods of time during hypoxia. Rat glioma cellsexposed to hypoxic cell culture conditions exhibiteda limitation on PH enzymatic activity accompanied byelevations in PHD2 mRNA, which peaked during the

Table 1.

Function Genes References

a. HRE-Regulated Genes

Iron uptake TfR [73]

Iron transport Tf [99]

Iron oxidation Cp [134]

Erythropoiesis Epo [69]

Heme catabolism HO-1 [72]

Angiogenesis VEGF, iNOS [135, 136]

Glucose metabolism GLUT1, GAPDH [137, 138]

Apoptosis NIP3, NIX [139]

Neurotransmitter synthesis TH [140]

b. IRE-Regulated Genes

Iron uptake TfR, DMT-1 [141]

Iron storage H- and L-Ft [142, 143]

Iron export IREG1 [144]

TCA cycle m-Aconitase [145]

Heme synthesis ALAS2 [146]

ALAS2, erythroid 5-aminolevulinate synthase; Cp, ceruloplasmin; DMT-1, divalent metal transporter-1; Epo, erythropoietin; Ft, ferritin; GAPDH,glyceraldehyde-3-P-dehydrogenase; GLUT1, glucose transporter 1; HO-1, heme oxygenase-1; iNOS, inducible nitric oxide synthase; NIP3; NIX; Tf, transferrin;TfR, transferrin receptor; TH, tyrosine hydroxylase; VEGF, vascular enthothelial growth factor.


potentiated GSH-depletion-induced cytotoxicity,while siRNA to HIF-1α reduces cell death associatedwith GSH depletion. The authors suggested thatperhaps the differential effects of HIF-1 activationare dependent on the redox state of the cell. Theactivation of HIF-1α has been associated with theinduction of several pro-apoptotic genes [110],therefore it is expected that a depletion ordestabilization of HIF-1α would result in protectionfrom cell death. However, there are many studies toconclude otherwise. For instance, anti-sensedepletion of HIF-1α was demonstrated to induce andaccelerate the apoptotic pathway in a glioma cell line[111]. Elevation in HIF-1α was also shown to preventapoptosis normally observed in sympathetic neuronsthat were NGF-deprived [112]. Contrary to thesefindings, decreases in neuronal damage induced byhypoxia or carotid artery occlusion were observed inmice with brain specific- late-stage deletion of HIF-1α[113]. It should therefore not be surprising that,depending on the type of injury stimuli, not all thesame genes are induced in different cell types as aresult of the diversity of HIF-1-mediated regulation.

dopamine and behavioral deficits triggered by 6-hydroxydopamine exposure [119]. The efficacy ofDFO, however, is limited due its inability to cross theblood brain barrier freely. Subsequently, otherchelators such as clioquinol (CQ) have beenidentified as more hydrophobic and efficacious inchelating both ferrous and ferric iron [120,121].Numerous studies have been published heraldingthe use of iron chelation in the treatment ofneurodegenerative disorders [122-126]. DFO, byvirtue of chelating iron required for prolyl hydroxylaseactivity, activate HIF-1α and has been demonstratedto prevent neuronal death in in vitro and in vivomodels of ischemia [127] and glucose deprivation[128]. DFO preconditioning in C6 astroglial cells hasbeen found to activate the HIF-1 pathway andprevent cell death normally associated withmitochondrial toxicants 3-nitroprionic acid, amitochondrial complex II inhibitor, and antimycin D, amitochondrial complex III inhibitor [129].

4b. Ferritin

Recently, our laboratory has shown that an over-expression of ferritin protects DAergic neurons of theSN against the effects of MPTP in young adult mice[123]. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) is a pro-toxicant which causes selectivedestruction of the SN in humans and other primates,resulting in an acute Parkinsonism and is widelyused as a model for the disease [130]. It has beendemonstrated that MPTP exerts its neurotoxic effectvia a selective inhibition of mitochondrial complex Iactivity, resulting in a reduction in ATP synthesis andreactive oxygen species accumulation [131,132]. Itremains to be determined, however, the exactmechanism involved in the ability of ferritinexpression to protect SN DAergic neurons from theneurotoxic effects of MPTP in young ferritin over-expressing mice. It may be mediated directly by areduction in the availability of free iron to participatein deleterious oxidative reactions or it could be dueto an indirect mechanism involving disruption of astress response pathway resulting from a decreasein the amount of iron needed as a cofactor forparticular enzymes in the pathway. In this case,chelation of iron by ferritin over-expression in DAergiccells could be acting via inhibition of PH activity,resulting in the subsequent activation of HIF-1α anddownstream activation of various genes as illustratedin Fig. (1b). The HIF-mediated transcription in HO-1may, for example, contribute to the neuroprotectionobserved in young ferritin transgenic mice exposedto MPTP. Short-term iron chelation can prevent theactivation of PH, allowing HIF-1 to activate severalprotective cellular factors in preventingneurodegeneration. Chronic iron chelation, however,could result in a sustained process of HIF-1activation that actually ends up bringing more ironinto the cell, overwhelming the cell’s normal capacityto deal with excess iron and to combat the additionaloxidative stress that occurs as a result of increasediron levels. While short-term iron chelation therapy

Some of the prominent features of aging areassociated with death of cells resulting from aninability to respond to stress. For example, there isoften a reduction in the levels of antioxidantenzymes, accumulation of mitochondrial DNA andoxidative damage [114,115], and increases inprotein degradation [116] with aging. Earlier studiesin aged mice have demonstrated impairments in thebinding of the HIF-1 complex to HREs on thevascular endothelial growth factor (VEGF) gene[117], suggesting this could be an underlying reasonwhy there is a compromised response to hypoxiasuch as decreased angiogenesis in the agedpopulation. Subsequent studies determined thatelevations in PHD3 protein expression wascorrelated to decreases in HIF-1α in human andmouse heart [118]. Recently, a group of structurallydiverse PHD inhibitors were used to determine theprotective effects of stabilizing HIF-1α in oxidativeand ischemic injury [105]. The inhibitors analyzedeither chelated iron (DFO or proprietary compound Afrom Fibrogen, South San Francisco, CA) orsequestered 2-oxoglutarate and ascorbate, bothimportant cofactors for PH activity. The investigatorsobserved a significant reduction in toxicity and ofinfarct volume associated with glutathione depletionand cerebral artery occlusion, respectively. Theseresponses, observed in both in vitro and in vivosystems, were accompanied by increased stability ofHIF-1α levels and HIF-mediated induction of VEGFand EPO. These studies further implicate thetherapeutic aspects of inhibiting PH in stabilizingcellular HIF-1α levels.

4a. Pharmacological Iron Chelators

The neuroprotective effects of iron chelation in aParkinson’s disease model was initially described instudies whereby intraventricular injection ofdesferoxamine (DFO) attenuated the depletion of


may be useful, its timing likely needs to be controlledso that appropriate levels of iron are available foressential cell functions while minimizing the oxidativehavoc that iron can wreck on the cell.

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Received: June 29, 2006 Revised: August 03, 2006 Accepted: August 04, 2006

Role of HIF-1 in Iron Regulation

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